The MAGE (Melanoma antigen) family of genes was originally identified due to the recognition from cytolytic lymphocytes derived from blood lymphocytes of cancer patients (Van der Bruggen et al 1991). The MAGE gene family now comprises over 20 members and is made up of MAGE A, B, C and D genes (Scanlan et al., (2002) Immunol Rev. 188:22-32; Chomez et al., (2001) Cancer Res. 61(14):5544-51). They are clustered on chromosome X (Lucas et al., 1998 Cancer Res. 58:743-752; Lucas et al., 1999 Cancer Res 59:4100-4103; Lucas et al., 2000 Int J Cancer 87:55-60; Lurquin et al., 1997 Genomics 46:397-408; Muscatelli et al., 1995 Proc Natl Acad Sci USA 92:4987-4991; Pold et al., 1999 Genomics 59:161-167; Rogner et al 1995 Genomics 29:725-731), and have a yet undefined function (Ohman et al. 2001 Exp Cell Res. 265(2):185-94). The MAGE genes are highly homologous and the members of the MAGE-A family, especially, have between 60-98% homology. MAGE genes are not expressed in all normal cells with the exception of expression in spermatogonia and placenta (Haas et al. 1988 Am J Reprod Immunol Microbiol 18:47-51; Takahashi et al. 1995 Cancer Res 55:3478-382).
The re-activation of MAGE gene expression in cancer is due to abnormal de-methylation of the promoter (De Smeet et al. 1996 Proc Natl Acad Sci USA 93(14):7149-53; De Smeet et al. 1999 Mol Cell Biol. 19(11):7327-35). The 12 MAGE-A genes are variably over-expressed in the following cancers: transitional-cell carcinoma, oesophageal carcinoma, melanoma, bladder and non-small cell lung carcinoma (NSCLC) (Scanlan et al. 2002 Immunol Rev. 188:22-32). The over expression and specificity of MAGE expression in cancerous tissues has led the MAGE-A3 protein to be used as an antigen for cancer vaccines (Scanlan et al. 2002 Immunol Rev. 188:22-32). However, due to the wide range of expression found in cancer patients, the level of expression of MAGE-A3 must be accurately estimated in each patient to direct the vaccination towards patients expressing the protein. MAGE-A3 is often referred to interchangeably as MAGE-3; both are used herein.
Melanoma
Patients presenting with malignant melanoma in distant metastasis (stage IV according to the American Joint Committee on Cancer (AJCC) classification) have a median survival time of one year, with a long-term survival rate of only 5%. Even the standard chemotherapy for stage IV melanoma has therapeutic response rates of only 8-25%, but with no effect on overall survival. Patients with regional metastases (stage III) have a median survival of two to three years with very low chance of long-term survival, even after an adequate surgical control of the primary and regional metastases (Balch et al., 1992 Semin Surg Oncol. 8(6):400-14). Most patients with stage I to III melanoma have their tumour removed surgically, but these patients maintain a substantial risk for relapse. Thus there remains a need to prevent melanoma progression, and to have improved treatment regimes for metastatic melanoma and adjuvant treatments for patients having had a primary tumour removed.
Lung Cancer
There are two types of lung cancer: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The names simply describe the type of cell found in the tumours. NSCLC includes squamous-cell carcinoma, adenocarcinoma, and large-cell carcinoma and accounts for around 80% of lung cancers. NSCLC is hard to cure and treatments available tend to have the aim of prolonging life as far as possible and relieving symptoms of disease. NSCLC is the most common type of lung cancer and is associated with poor outcomes. Of all NSCLC patients, about 25% have loco-regional disease at the time of diagnosis and are still amenable to surgical excision (stages IB, IIA or IIB according to the AJCC classification). However, more than 50% of these patients will relapse within the two years following the complete surgical resection. There is therefore a need to provide better treatment for these patients.
MAGE-A3 Expression
Previously a number of methods have attempted to measure the expression of MAGE-A3 genes within both cell lines and tumour samples. Semi-quantitative RT-PCR (De Plaen et al. 1994 Immunogenetics 40(5):360-9), other PCR based techniques and also low-density microarray have all been used (Zammatteo et al. 2002 Clinical Chemistry 48(1) 25-34). However in many of these studies a major problem has been the very high homology between the MAGE family members causing false positives. For large Phase II and III trials a quantitative high throughput assay, capable of specifically identifying MAGE-A3-expressing samples and of reducing the likelihood of samples falsely testing-positive, is desirable.
Another difficulty arises with the use of Formalin-Fixed, Paraffin-Embedded (FFPE) tumour tissue, which is the usual method of tumour tissue preservation within clinical centres. The fixation in formalin changes the structure of molecules of RNA within the tissue, causing cross linking and also partial degradation. The partial degradation leads to the creation of smaller pieces of RNA of between 100-300 base pairs. These structural changes to the RNA make it difficult to use RNA extracted from FFPE tissue in conventional diagnostic techniques.